pcr: amplified to the end point · events: the polymerase chain reaction “the idea first came to...

PCR AMPLIFIED TO THE END POINT

Page 3A Series of Fortunate Events The Polymerase Chain Reaction

Page 4PCR World Applications

Page 5Amplification Angst Overcoming Common PCR Challenges

Page 6Fatest to the Finish Line Increasing Throughput

Custom Publishing From Sponsored By

INTRODUCING 2-MINUTE PCRSince PCR was introduced DNA replication has incrementally gotten easier and faster Now a revolution has happened NEXTGENPCR heats and cools instantly mdash ldquoramp ratesrdquo no longer exist Using one thermocycler you can run more than ten plates per hour The math adds up quickly mdash 8 hours of 10 plates in a 384-well format means more than 30000 data points in a single day

GO FASTERwwwcanon-biomedicalcomgofaster

contactuscanon-biomedicalcom | wwwcanon-biomedicalcom | 844-CANONBIO

Ultrafast100 base pairs and 30 cycles in 2 minutes

Versatile Open chemistry

Flexible 96-well or 384-well format without changing blocks

PreciseWell uniformity of less than 01degC

For research use only Not for use in diagnostic proceduresThe NEXTGENPCR products are manufactured by Molecular Biology Systems BV wwwnextgenpcrcomAll referenced product names and other marks are trademarks of their respective owners copy 2018 Canon BioMedical Inc All rights reserved

TheScientist 2018 the-scientistcom3


A Series of Fortunate Events The Polymerase Chain ReactionldquoThe idea first came to Mullis in 1983 while he was driving down a California highway a pivotal moment that has since transformed life sciencerdquo

PCRrsquos Humble Beginnings

The polymerase chain reaction (PCR) was developed by Nobel Laureate Kary B Mullis in 1985 while he was working as a molecular biologist at the Cetus Corporation (a biotechnology firm in Emeryville California) to make millions of copies of small amounts of or hard-to-come-by DNA1 The idea first came to Mullis in 1983 while he was driving down a California highway a pivotal moment that has since transformed life science PCR was developed as a manual process initially which meant it was slow and laborious 23 Seeing its potential for many areas of research scientists at the Cetus Corporation partnered with PerkinElmer and set about to automate the process and thus ldquoMr Cyclerdquo the worldrsquos first thermocycler was born By 1989 Cetus and Hoffman-LaRoche had joined forces to develop and commercialize in vitro human diagnostic products using PCR technology4 Several commercial companies have since picked up the baton resulting in an abundance of choices for life scientists

PCR in the World

PCR is now a staple in biochemistry biology and chemistry labs worldwide In research itrsquos primarily used as a starting point for various other assays Examples include creating multiple copies of DNA before sequencing or before introducing DNA into a host organism5 However PCR has become an essential assay in the real world and helped to advance research in areas as diverse as diagnosing Legionnairersquos disease6 and detecting HIV7 The technique is crucial for the diagnosis of genetic disease itrsquos used in preimplantation diagnosis prior to in vitro fertilization procedures screening fetuses for genetic disease before birth and diagnosing inherited diseases8 It can be used for precision medicine such as in the treatment of cancer9 and itrsquos also used to monitor the spread of infectious diseases in both animal and human populations10 PCR can even be used in archaeology to identify insects trapped in amber11 or to track human migration patterns12

Steps to Extension The Basic PCR Procedure

The fundamental steps of a PCR reaction are denaturation annealing and extension and these steps are repeated for many cycles A thin PCR tube or plate containing a DNA template free nucleotides and two short DNA primers are mixed together along with a DNA polymerase enzyme and magnesium-containing buffer The tube is heated (typically 94 degC to 96 degC) to denature the DNA template into single strands exposing the DNA segment of interest Upon cooling (typically 45 degC to 60 degC) the DNA primers anneal to complementary DNA sections The tube is then heated to the active temperature of the DNA polymerase (typically to 72 degC) extending the primers by incorporating the free nucleotides into the complementary strand of the DNA In traditional end-point PCR one cycle takes a few minutes and results in two copies of the DNA segment of interest This cycle is repeated (25 to 45 cycles) to create millions of copies of the desired DNA fragment5

Take Your Pick Types of PCR

End-point PCR describes analysis after all cycles of PCR are complete (ie after amplification is complete) It is the name for the original standard amplification technique but PCR now comes in many varieties Real-time PCR (also known as quantitative PCR qPCR or RT-qPCR) measures the accumulation of amplified DNA in real time which allows quantitative analysis of gene expression removes the need to run PCR product on a gel after the reaction and gives insight into how well a reaction worked Reverse-transcription PCR (RT-PCR) uses RNA as a template prior to being converted to cDNA for the PCR reaction which is a sensitive technique for genetic disease testing characterizing gene expression and detecting viruses Nested PCR is a modified PCR protocol whereby non-specific binding is reduced by using two pairs of PCR primers (one outer pair and one more precise nested pair) in two consecutive rounds of PCR to enhance the specificity and yield of the desired amplicon Multiplex PCR amplifies multiple targets in a single PCR reaction which saves time and also makes comparison of multiple amplicons possible13

For references please see page 7



ldquoPCR and its inherent sensitivity have revolutionized genetic testingrdquo

PCR World Applications

PCR is the cornerstone of genetic diagnostic testing however it is also the foundation for various other applications in scientific fields as diverse as forensics and agriculture

PCR Proof Genetic Diagnostics

Cystic fibrosis sickle cell anemia phenylketonuria and muscular dystrophy are just a handful of the genetic diseases that use PCR as a test method1 Genetic testing is normally performed on cells or sometimes just a single cell taken from an embryo or fetus before birth PCR and its inherent sensitivity have revolutionized genetic testing in this respect and have allowed individuals undergoing in vitro fertilization to test for certain genetic diseases before an embryo is even implanted2 Paternity testing is also carried out by PCR A cheek swab is taken from the parents and child and sections of DNA called loci are amplified to enable the comparison between each individual3

Solving Crimes Forensics

Genetic fingerprinting is a tool used for identifying or ruling out suspects in criminal cases based on their DNA profile Minute amounts of DNA from samples as diverse as hair touch DNA or bodily fluids can be taken from a crime scene and amplified by PCR identifying a single suspect from millions of others 4 DNA databases allow the comparison of all known genetic profiles PCR is the gold standard in genetic fingerprinting as its fast turnaround small equipment footprint ease of use and high sensitivity allow for simple yet effective analysis of the smallest traces of DNA PCR from mitochondrial genes has also gained popularity in recent years in forensic science due to mitochondriarsquos inherent high copy number per cell matrilineal inheritance and lack of recombination of its DNA4

A Tool in Agriculture

Agriculture makes use of PCR during product development seed quality control gene discovery and cloning vector construction transformant identification screening and characterization are just a few uses Third-party diagnostic testing services rely on PCR to test for the presence or quantity of genetically modified (GM) material in a product and to estimate GM copy number or zygosity in seeds and plants5

In grain handling and processing GM events are also tested for by

PCR for example a country producing GM crops may export to a country that does not allow certain genetic modifications In this case the importing country will use qualitative PCR to test for GM events to make sure the imported product is compliant5

Sequencing

Traditional (Sanger) sequencing and next-generation sequencing (NGS) both make full use of PCR to amplify DNA prior to DNA sequencing and to create templates for sequencing Sanger sequencing involves using capillary electrophoresis in combination with automated base calls to sequence long DNA fragments producing one forward and reverse read whereas NGS allows millions of fragments to be sequenced in a single run To avoid problems with DNA strand reassociation during sequencing reactions single-stranded DNA (ssDNA) templates may be produced by PCR6

All the Small Things PCR in Microbiology

Rapid detection is essential for foodborne illnesses to enable the correct treatment protocol to be followed PCRs sensitivity and specificity make it an ideal tool in this respect Various PCR types are used for pathogen detection including traditional PCR and quantitative PCR which can detect single bacterial species such as Ecoli and Shigella spp and multiplex PCR which can detect multiple species at once through simultaneous amplification of multiple gene targets7

PCR also has use in the beer industry The technique is used to detect bacterial contamination of wort beer and yeast slurries or to detect the strain health and purity of yeast cultures Real-time PCR quickly provides brewers with valuable information about the quality of their product that previously took days to obtain8

Its Hereditary Genealogy and PCR

A particularly useful aspect of PCR is its ability to amplify specific sections of the Y chromosome and mitochondrial DNA which are used in genealogy testing Both have been used extensively to study human lineages as they are both inherited in a haploid manner9


Ever had to deal with a petulant DNA template shady PCR product smearing or mysterious extra bands on your gels PCR is full of challenges that can leave you feeling left in the dark Our troubleshooting

guide will help guide you back to the light

AMPLIFICATION ANGST

NON-SPECIFIC AMPLIFICATION Potential causes Premature replication

annealing temperatures too low contamination poor primer design incorrect magnesium

concentration

Set up the PCR experiment on ice use a hot start polymerase increase the annealing temperature use a dedicated clean work area and pipette use a longer primer avoid GC-rich ends check for complementary sequences adjust the magnesium concentration in small increments

LOW YIELD Potential causes Poor primer design incorrect

annealing temperature thick-walled PCR tubes incorrect primer concentration not enough

cycles low starting DNA concentration

Optimize the primer using software optimize the annealing temperature by 1ndash2 degC use thin-walled PCR tubes increase the primer concentration in increments up to 05 microM increase cycling up to 45 cycles start with a DNA sample in the ngul range

INCORRECT PRODUCT SIZE Potential causes Nuclease contamination poor

primer design incorrect annealing temperature

Use fresh solutions optimize the primer optimize the annealing temperature by 1ndash2 degC

SMEARING OF PCR PRODUCT Potential causes DNA starting template

concentration too high contamination DNA polymerase concentration too high too

many cycles poor primer design incorrect magnesium concentration

Determine the best starting concentration using serial dilutions use fresh solutions and a clean work area lower the DNA polymerase concentration reduce the cycle number in 3-cycle increments optimize the primer optimize the magnesium concentration

SEQUENCE ERRORS Potential causes Incorrect nucleotide

concentration damaged DNA template sequence toxicity to the host problems with

reaction conditions and components

Use a fresh nucleotide mixture or DNA template limit UV exposure clone into a different vector decrease the magnesium concentration extension time and number of cycles try using a high-fidelity polymerase

NO PRODUCT Potential causes Almost anything Primer

problems missing reaction components incorrect temperatures during PCR suboptimal

template contamination

Change the PCR conditions one component at a time to troubleshoot (optimize primer fresh reagents optimize DNA template optimize magnesium concentration increase cycles check thermocycler program)

O V E R C O M I N G C O M M O N P C R C H A L L E N G E S

END-POINT PCR SCIENCErsquoS STARTING BLOCKS

PREDICAMENT SOLUTIONS



Fastest to the Finish Line Increasing Throughput

An avalanche of publications since the 1980s exists that details improvements to PCR protocols and components Despite these improvements a typical PCR reaction

still takes hours to complete However the tides are changing and things look set to speed up

Time is of the Essence PCR Problems

Itrsquos an unfortunate fact that any type of laboratory analysis comes with its fair share of challenges and PCR is no exception Potential issues include contamination sequence errors thermocycler problems DNA template and primer issues low yields or no bands whatsoever - the source of many a researcherrsquos hair pulling1 Many of these issues can be overcome by consecutively changing one reaction component at a time Often magnesium or DNA polymerase concentrations need adjustment a fresh batch of reagents is required or thermocycler conditions necessitate modification2 Troubleshooting PCR problems takes time and when a single PCR reaction takes hours to complete time is of the essence However all of these problems can be overcome and with recent advancements in PCR technology the troubleshooting process is quicker than ever before

The Collaboration Game PCR Progression

Scientists from commercial government and university research laboratories are to thank for advancements in PCR technology over the past 35 years One of the first major PCR advancements in 1986 was the isolation of the thermostable Taq polymerase from Thermus aquaticus3 a bacterium that resides in hot springs which removed the need for human involvement during PCR (DNA polymerase from E coli was used prior to this discovery) Taq has remained the DNA polymerase of choice for PCR ever since and further enzyme modifications enable amplification of very long strands of DNA or copying with very high fidelity

Computer software revolutionized PCR further making once difficult-to-solve DNA primer and template problems much easier to deconvolute4 Furthermore the creation of and continual addition to genetic libraries such as GenBank5 and EMBL6 have simplified the development and validation of molecular-based diagnostic procedures using PCR

Equipment advances since the first automated thermocycler include new robotic elements microfluidics chip technology thin-walled tubes and nanotechnology These technological breakthroughs have made PCR faster workflows more efficient and allowed the development of different types of PCR such as quantitative and reverse-transcription PCR Other technical advances include ldquohot startrdquo systems and the miniaturization of PCR assays7 These advances would not have happened if it werent for multiple successful collaborations between industry and academia

Full Speed Ahead Ultrafast PCR

Traditionally researchers using PCR were from academic laboratories But more recently the industry is seeing a trend where biopharmaceutical and other commercial ventures are also requesting PCR equipment And with the increasing demand for PCR comes the need for highly efficient thermocyclers able to perform PCR in the shortest amount of time possible able to handle multiple samples at once and able to do all of this without compromising assay integrity

Ultrafast PCR is the one of most recent advancements in PCR New technology allows the use of microplates that can handle up to 384 samples while eliminating temperature ramping requirements enabling PCR reaction times of 2 to 10 minutes8

This increased speed provides the flexibility to do high-throughput work or simply get to the result much faster to move research forward

This increase in PCR speed can not only quicken the now-routine technique that has become a staple method in life science labs throughout the globe but also make the tedious task of PCR troubleshooting a little less letrsquos say traumatic

From industry to agriculture diagnostics to microbiology genealogy to forensics ultrafast PCR is a simple solution to increase productivity With advancements in PCR happening at a lightening-fast rate it will be interesting to see where this California-dreamed-up invention takes us next


New technology allows the use of microplates that can handle up to 384 samples while eliminating temperature ramping requirements enabling PCR reaction times of 2 to 10 minutes



Article 1 - A Series of Fortunate Events The Polymerase Chain Reaction

References1 ldquoKary B Mullis ndash Factsrdquo Nobelprizeorg Nobel Media AB 2014

Accessed on June 11 20182 RK Saiki et al ldquoA Novel Method for the Prenatal Diagnosis of Sickle

Cell Anemiardquo Amer Soc Human Genetics 19853 RK Saiki et al ldquoEnzymatic amplification of β-globin genomic

sequences and restriction site analysis for diagnosis of sickle cell anemiardquo Science 2301350-1354 1985

4 M Fakruddin et al ldquoCompetitiveness of polymerase chain reaction to alternate amplification methodsrdquo Am J Mol Biol 371-80 2013

5 L Garibyan and N Avashia ldquoResearch techniques made simple Polymerase chain reaction (PCR)rdquo J Invest Dermatol 133(3)e6 2013

6 BMW Diederen et al ldquoUtility of real-time PCR for diagnosis of Legionnairesrsquo disease in routine clinical practicerdquo J Clin Microbiol 46(2)671-677 2008

7 M Holodniy et al ldquoDetection and quantification of Human Immunodeficiency Virus RNA in patient serum by use of the polymerase chain reactionrdquo J Infect Dis 163(4)862-866 1991

8 W Navidi and N Arnheim ldquoUsing PCR in preimplantation genetic disease diagnosisrdquo Hum Reprod 6(6)836-849 1991

9 L Chin et al ldquoCancer genomics from discovery science to personalized medicinerdquo Nat Med 17297-303 2011

10 ND Wolfe et al ldquoOrigins of major human infectious diseaserdquo Nature 447279-2832007

11 RJ Cano et al ldquoAmplification and sequencing of DNA from a 120-135-million-year-old weevilrdquo Nature 363536-538 1993

12 MC Fisher et al ldquoBiogeographic range expansion into South America by Coccidioides immitis mirrors New World patterns of human migrationrdquo PNAS 98(8)4558-4562 2001

13 J Singh et al ldquoA critical review on PCR its types and applicationsrdquo Int J Adv Res Biol Sci 165-80 2014

Article 2 - PCR World Applications

References1 N Mahdieh and B Rabbani ldquoAn overview of mutation detection

methods in genetic disordersrdquo Iran J Pediatr 23(4)375-388 20132 Y-S Yang et al ldquoPreimplantation genetic screening of blastocysts

by multiplex qPCR followed by fresh embryo transfer validation and verificationrdquo Mol Cytogenet 849-55 2015

3 MA Jobling et al ldquoThe Y chromosome in forensic analysis and paternity testingrdquo Int J Legal Med 110(3)118-124 1997

4 A Alonso et al ldquoReal-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studiesrdquo Forensic Sci Int 139(2-3)141-149 2004

5 M Lipp et al ldquoPolymerase chain reaction technology as analytical tool in agricultural biotechnologyrdquo J AOAC Int 88(1) 136-155 2005

6 UB Gyllensten ldquoPCR and DNA sequencingrdquo Biotechniques 7(7) 700-708 1989

7 JW-F Law et al Rapid ethods for the detection of foodborne bacterial pathogens principles applications advantages and limitations Front Microbiol 5(770)1-19 2014

8 K Sakamoto and WN Konings Beer spoilage bacteria and hop resistance Int J Food Microbiol 89(2-3)105-124 2003

9 J Cummins Mitochondrial DNA and the Y chromosome parallels and paradoxes Reprod Fertil Dev 13(7-8)533-542 2001

Article 3 - Fastest to the Finish Line Increasing Throughput

References1 SA Bustin et al ldquoQuantitative real-time RT-PCR ndash a perspectiverdquo J

Mol Endocrinol 34597-601 20052 KH Roux ldquoOptimization and troubleshooting in PCRrdquo Adapted from

PCR Primer A Laboratory Manual 2nd edition (eds Dieffenbach and Dveksler) CSHL Press Cold Spring Harbor NY USA 2003

3 RK Saiki et al ldquoPrimer-directed enzymatic amplification of DNArdquo Science 239487-491 1988

4 KA Abd-Elsalam ldquoBioinformatic tools and guideline for PCR primer designrdquo Afr J Biotechnol 2(5)91-95 2003

5 DA Benson ldquoGenBankrdquo Nucleic Acids Res 36D25-D30 2008 6 M Goujon et al ldquoA new bioinformatics analysis tool framework at

EMBL-EBIrdquo Nucleic Acids Res 38(2)W695-W699 20107 LH Lauerman ldquoAdvances in PCR technologyrdquo Anim Health Res Rev

5(2)247-248 2004 8 SA Bustin ldquoHow to speed up the polymerase chain reactionrdquo

Biomolecular Detection and Quantification 1210-14 2017

LEARN MOREwwwcanon-biomedicalcomgofastercontactuscanon-biomedicalcom | 844-CANONBIO

For research use only Not for use in diagnostic procedures The NEXTGENPCR products are manufactured by Molecular Biology Systems BV wwwnextgenpcrcom

All referenced product names and other marks are trademarks of their respective owners copy 2018 Canon BioMedical Inc All rights reserved

PCR AMPLIFICATION OF A 100 BP DNA FRAGMENT IN LESS THAN 2 MINUTES

NEXTGENPCR a novel thermocycler from Molecular Biology SystemsNEXTGENPCR virtually eliminates PCR ramp times by using unique thermal zones As illustrated in Figure 1 a microplate moves between thermal zones maintaining the temperatures to denature anneal and extend DNA The wells of the microplate are made from 40 micron polypropylene and the samples are heat sealed into individual wells Compression of the ultrathin microplate wells by the thermal zone blocks instantly transfers the applicable temperature and mixes the sample By optimizing each step of the PCR DNA amplification can be as quick as 1 minute and 59 seconds as demonstrated by the amplification of a 100 bp fragment of the NQ01 gene shown in Figure 2

NEXTGENPCR Technology

Microplate moves to the thermal zone

DenatureExtendAnneal

Heat blocks close for optimal heat transfer and mixing

Microplate moves to the next thermal zone

Reaction mix Cycling profile

1X PCR buffer 03 mM of each dNTP 4 mM MgCl2 25 ng template DNA 1 M of each primer 05 Units of KAPA2G Fast HotStart DNA Polymerase

Initial Denaturation 98degC 10 sec

5 cycles Denaturation 98degC 2 secAnnealing 60degC 2 secExtension 75degC 2 sec


Figure 2 NQ01 amplification product

Figure 1 Diagram of NEXTGENPCR thermal zones

ULTRAFAST PRECISE FLEXIBLE UNCOMPLICATED VERSATILE

Amplification of a 100 bp fragment of the NQ01 gene Using the advanced mode function of NEXTGENPCR and reagents from KAPA Biosystems the 100 bp fragment was amplified then visualized by running on a 2 agarose gel next to a 100 bp DNA ladder

BASE PAIRS STEPS

Denature

Anneal

Extend

CYCLESMINUTES

INTRODUCING 2-MINUTE PCRSince PCR was introduced DNA replication has incrementally gotten easier and faster Now a revolution has happened NEXTGENPCR heats and cools instantly mdash ldquoramp ratesrdquo no longer exist Using one thermocycler you can run more than ten plates per hour The math adds up quickly mdash 8 hours of 10 plates in a 384-well format means more than 30000 data points in a single day

GO FASTERwwwcanon-biomedicalcomgofaster

contactuscanon-biomedicalcom | wwwcanon-biomedicalcom | 844-CANONBIO

Ultrafast100 base pairs and 30 cycles in 2 minutes

Versatile Open chemistry

Flexible 96-well or 384-well format without changing blocks

PreciseWell uniformity of less than 01degC

For research use only Not for use in diagnostic proceduresThe NEXTGENPCR products are manufactured by Molecular Biology Systems BV wwwnextgenpcrcomAll referenced product names and other marks are trademarks of their respective owners copy 2018 Canon BioMedical Inc All rights reserved






PCR in the World




















Sequencing










AMPLIFICATION ANGST



concentration




































































































BASE PAIRS STEPS

Denature

Anneal

Extend

CYCLESMINUTES






PCR in the World




















Sequencing










AMPLIFICATION ANGST



concentration




































































































BASE PAIRS STEPS

Denature

Anneal

Extend

CYCLESMINUTES














Sequencing










AMPLIFICATION ANGST



concentration




































































































BASE PAIRS STEPS

Denature

Anneal

Extend

CYCLESMINUTES



AMPLIFICATION ANGST



concentration




































































































BASE PAIRS STEPS

Denature

Anneal

Extend

CYCLESMINUTES













































































BASE PAIRS STEPS

Denature

Anneal

Extend

CYCLESMINUTES

pcr: amplified to the end point · events: the polymerase chain reaction “the idea first came to...

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